1. Field of the Invention
2. Background of the Invention
2.1 Liposomes
2.2 Water-Soluble Sterols
3. Summary of the Invention
4. Brief Description of the Figures
5. Detailed Description of the Invention
6. Example: Cholesterol Hemisuccinate Liposomes Entrapping Water-soluble Compounds
6.1 Liposomes Prepared Using Various Salts Forms of Cholesterol Hemisuccinate
6.1.1 Tris-Salt Cholesterol Hemisuccinate MLVs
6.1.2 2-Amino-2-Methyl-1, 3-Propanediol Cholesterol Hemisuccinate-MLVs
6.1.3 2-Aminoethanol Cholesterol Hemisuccinate-MLVs
6.1.4 Bis-Tris-Propane Cholesterol Hemisuccinate-MLVs
6.1.5 Triethanolamine Cholesterol Hemisuccinate-MLVs
6.1.6 Miconazole Cholesterol Hemisuccinate-MLVs
6.1.7 Cholesterol Hemisuccinate-SUVs Prepared by Sonication
6.1.8 Cholesterol Hemisuccinate-SUVs Prepared by Extrusion Techniques
6.1.9 Miconazolexe2x80x94CHS-Tris Cream
6.1.10 Terconazolexe2x80x94CHS-Tris Cream
6.1.11 Miconazolexe2x80x94CHS-Tris Suppository
6.1.12 In Vivo Activity for Vaginal Candida Infections
6.2 Entrapment of Inulin in Cholesterol Hemisuccinate MLVs
6.2.1 Encapsulation Efficiency of Inulin in Cholesterol Hemisuccinate-MLVs and Egg Phosphatidylcholine-MLVs
6.3 Entrapment of Inulin in Cholesterol Hemisuccinate SUVs
6.4 Entrapment of Chromium in Cholesterol Hemisuccinate MLVs
6.4.1 Encapsulation Efficiency of Chromium in Cholesterol Hemisuccinate-MLVs
6.4.2 Captured Volume in Cholesterol Hemisuccinate MLVs: Chromium Entrapment Cholesterol Hemisuccinate Concentration
6.5 Ultrastructure of Cholesterol Hemisuccinate Liposomes
6.6 X-Ray Diffraction Analysis of Cholesterol Hemisuccinate Liposomes
6.7 Electron Spin Resonance Analysis of Cholesterol Hemisuccinate Liposomes
6.8 Isotonic Swelling of Cholesterol Hemisuccinate Liposomes
7. Example: Cholesterol Hemisuccinate Liposomes Entrapping Sparingly Soluble Compounds
7.1 Bovine Growth Hormone Entrapped in Cholesterol Hemisuccinate-SUVs
7.2 Insulin Entrapped in Cholesterol Hemisuccinate SUVs
7.3 Tylosin Entrapped in Cholesterol Hemisuccinate-SUVs
8. Example: The use of Cholesterol Hemisuccinate Liposomes to Entrap Lipid Soluble Compounds
8.1 Indomethacin Entrapped in Cholesterol Hemisuccinate-MLVs
8.1.2 Ultrastructure of Cholesterol Hemisuccinate Vesicles Containing Indomethacin
8.2 Diazepam Entrapped in Cholesterol Hemisuccinate-SUVs
9. Example: The Use of Cholesterol Hemisuccinate Liposomes to Determine Aminoglycoside Concentration in Serum
10. Example: In Vivo Administration of Cholesterol Hemisuccinate Liposomes
10.1 Treatment of Joint Arthritis Using Indomethacin Entrapped in Cholesterol Hemisuccinate-MLVs
10.2 In Vivo Administration of Diazepam Entrapped in Cholesterol Hemisuccinate SUVs
10.2.1 organ Distribution After Intravenous Innoculation
10.3 In Vivo Administration of Chromium Entrapped in Cholesterol hemisuccinate-MUVs
10.4 In Vivo Administration of Human Growth Hormone Entrapped in Cholesterol Hemisuccinate MLVs
The present invention relates to methods and compositions for the entrapment of compounds in liposomes composed of salt forms of organic acid derivatives of sterols that are capable of forming bilayers.
Sterols such as cholesterol or other lipids, to which a hydrophilic moiety such as a salt form of an organic acid is attached, can be used to prepare suspensions of multilamellar or small unilamellar vesicles. The sterol liposomes of the present invention may be prepared with or without the use of organic solvents. These vesicles may entrap water-soluble compounds, partially water-soluble compounds, and water-insoluble compounds.
The sterol vesicles described herein are particularly useful for the entrapment of biologically active compounds or pharmaceutical compounds which can be administered in vivo. Alternatively, the sterol liposomes of the present invention may be used in vitro. For instance, the cholesterol hemisuccinate liposomes described herein may be used in vitro in divalent cation-dependent assay systems.
Liposomes are completely closed bilayer membranes containing an encapsulated aqueous phase. Liposomes may be any variety of multilamellar vesicles (onion-like structures characterized by concentric membrane bilayers each separated by an aqueous layer) or unilamellar vesicles (possessing a single membrane bilayer).
Two parameters of liposome preparations are functions of vesicle size and lipid concentration: (1) Captured volume, defined as the volume enclosed by a given amount of lipid, is expressed as units of liters entrapped per mole of total lipid (1molxe2x88x921). The captured volume depends upon the radius of the liposomes which in turn is affected by the lipid composition of the vesicles and the ionic composition of the medium. (2) Encapsulation efficiency, defined as the fraction of the aqueous compartment sequestered by the bilayers, is expressed as a percentage. The encapsulation efficiency is directly proportional to the lipid concentration; when more lipid is present, more solute can be sequestered within liposomes. (See Deamer and Uster, 1983, Liposome Preparation: Methods and Mechanisms, in Liposomes, ed. M. Ostro, Marcel Dekker, Inc., NY, pp. 27-51.)
The original method for liposome preparation (Bangham et al., 1965, J. Mol. Biol. 13: 238-252) involved suspending phospholipids in an organic solvent which was then evaporated to dryness leaving a waxy deposit of phospholipid on the reaction vessel. Then an appropriate amount of aqueous phase was added, the mixture was allowed to xe2x80x9cswell,xe2x80x9d and the resulting liposomes which consisted of multilamellar vesicles (hereinafter referred to as MLVs) were dispersed by mechanical means. The structure of the resulting membrane bilayer is such that the hydrophobic (non-polar) xe2x80x9ctailsxe2x80x9d of the lipid orient toward the center of the bilayer while the hydrophilic (polar) xe2x80x9cheadsxe2x80x9d orient towards the aqueous phase. This technique provided the basis for the development of the small sonicated unilamellar vesicles (hereinafter referred to as SUVs) described by Papahadjopoulos and Miller (1967, Biochim. Biophys. Acta. 135: 624-638). Both MLVs and SUVs, however, have limitations as model systems.
In attempts to increase captured volume or encapsulation efficiency a number of methods for the preparation of liposomes comprising phospholipid bilayers have been developed; however, all methods require the use of organic solvents. Some of these methods are briefly described below.
An effort to increase the encapsulation efficiency involved first forming liposome precursors or micelles, i.e., vesicles containing an aqueous phase surrounded by a monolayer of lipid molecules oriented so that the polar head groups are directed towards the aqueous phase. Liposome precursors are formed by adding the aqueous solution to be encapsulated to a solution of polar lipid in an organic solvent and sonicating. The liposome precursors are then emulsified in a second aqueous phase in the presence of excess lipid and evaporated. The resultant liposomes, consisting of an aqueous phase encapsulated by a lipid bilayer are dispersed in aqueous phase (see U.S. Pat. No. 4,224,179 issued Sep. 23, 1980 to M. Schneider).
In another attempt to maximize the encapsulation efficiency, Papahadjopoulos (U.S. Pat. No. 4,235,871 issued Nov. 25, 1980) describes a xe2x80x9creverse-phase evaporation processxe2x80x9d for making oligolamellar lipid vesicles also known as reverse-phase evaporation vesicles (hereinafter referred to as REVs). According to this procedure, the aqueous material to be encapsulated is added to a mixture of polar lipid in an organic solvent. Then a homogeneous water-in-oil type of emulsion is formed and the organic solvent is evaporated until a gel is formed. The gel is then converted to a suspension by dispersing the gel-like mixture in an aqueous media. The REVs produced consist mostly of unilamellar vesicles (large unilamellar vesicles, or LUVs) and some oligolamellar vesicles which are characterized by only a few concentric bilayers with a large internal aqueous space.
Much has been written regarding the possibilities of using liposomes for drug delivery systems. See, for example, the disclosures in U.S. Pat. No. 3,993,754 issued on Nov. 23, 1976, to Yeuh-Erh Rahman and Elizabeth A. Cerny, and U.S. Pat. No. 4,145,410 issued on Mar. 20, 1979, to Barry D. Sears. In a liposome drug delivery system the medicament is entrapped during liposome formation and then administered to the patient to be treated. The medicament may be soluble in water or in a non-polar solvent. Typical of such disclosures are U.S. Pat. No. 4,235,871 issued Nov. 25, 1980, to Papahadjopoulos and Szoka and U.S. Pat. No. 4,224,179, issued Sep. 23, 1980 to M. Schngeider. When preparing liposomes for use in vivo it would be advantageous (1) to eliminate the necessity of using organic solvents during the preparation of liposomes; and (2) to maximize the encapsulation efficiency and captured volume so that a greater volume and concentration of the entrapped material can be delivered per dose.
A variety of sterols and their water soluble derivatives have been used for cosmetic, pharmaceutical and diagnostic purposes. Of the water soluble sterols, for example, branched fatty acid cholesterol esters, steroid esters and PEG-phytosterols have been used in cosmetic preparations (European Patent. Application No. 28,456; U.S. Pat. No. 4,393,044; and Schrader, Drug and Cosmetic Industry, September. 1983, p.33 and October 1983, p.46). Thakkar and Kuehn (1969, J. Pharm. Sci. 58(7): 850-852) disclose the solubilization of steroid hormones using aqueous solutions of steroidal non-ionic surfactants, specifically ethoxylated cholesterol (i.e., PEG-cholesterol) at a concentration of 1-5%. However, the effectiveness or utility of the solubilized steroid hormones in vivo was not demonstrated. A number of water soluble cholesterols have been prepared and used as water-soluble standards for the determination of cholesterol levels in biological fluids (U.S. Pat. No. 3,859,047; U.S. Pat. No. 4,040,784; U.S. Pat. No. 4,042,330; U.S. Pat. No. 4,183,847; U.S. Pat. No. 4,189,400; and U.S. Pat. No. 4,224,229). Shinitzky et al. (1979, Proc. Natl. Acad. Sci. USA 76:5313-5316) incubated tumor cells in tissue culture medium containing a low concentration of cholesterol and cholesteryl hemisuccinate. Incorporation of cholesterol or cholesteryl hemisuccinate into the cell membrane decreased membrane fluidity and increased membrane-lipid microviscosity.
Cholesterol and other sterols, have also been incorporated into phospholipid liposome membranes in order to alter the physical properties of the lipid bilayers. For example, in a recent abstract, Ellens et al. (1984, Biophys. J. 45: 70a) discuss the effect of H+ on the stability of lipid vesicles composed of phosphatidylethanolamine and cholesteryl hemisuccinate. In fact, Brockerhoff and Ramsammy (1982, biochim. Biophys. Acta. 691:227-232) reported that bilayers can be constructed which consist entirely of cholesterol, provided a stabilizing hydrophilic anchor is supplied. Multilamellar and unilamellar cholesterol liposomes were prepared in a conventional manner described above evaporating to dryness the cholesterol derivatives (i.e., cholesterol-phosphocholine, cholesterol-polyethylene glycol, or cholesterol- SO4) dispersed in an organic solvent leaving a lipid film deposited in the reaction vessel. The lipid films were sonicated under 2 ml water using an ultrasonic homogenizer with a microtip. Formation of multilamellar vesicles required 10 minutes sonication, whereas formation of small unilamellar vesicles required 4 hours of sonication. The resulting suspensions of multilamellar liposomes were milky whereas the suspensions of unilamellar liposomes were transparent.
However, the ability to efficiently entrap bioactive agents in sterol vesicles which are suitable for administration in vivo to provide for the administration of higher doses of water-soluble agents and to facilitate the administration of water-insoluble agents has not heretofore been explored.
The present invention involves methods and compositions for the entrapment of various compounds in liposomes, the bilayers of which comprise salt forms of organic acid derivatives of sterols. Entrapment of a compound is defined herein as the encapsulation of a water-soluble compound in the aqueous compartment of the liposome or the entrapment of a water-insoluble compound within the sterol bilayer. The tris(hydroxymethyl)aminomethane salt (tris-salt) form of organic acid derivatives of sterols are particularly useful as the vesicle bilayer ingredient.
The method for preparing the sterol vesicles involves adding to an aqueous buffer a salt form of an organic acid derivative of a sterol capable of forming closed bilayers in an amount sufficient to form completely closed bilayers which entrap an aqueous compartment. A suspension of multilamellar vesicles is formed by shaking the mixture. The formation of vesicles is facilitated if the aqueous buffer also contains the counterion of the salt in solution. Furthermore, if the dissociated salt form of the organic acid derivative of a sterol is negatively charged at neutral pH, the aqueous buffer should be essentially free of divalent or multivalent cations. Similarly, when the dissociated salt form of the organic acid derivative of a sterol is positively charged at neutral pH, the aqueous buffer should be essentially free of multivalent anions. The application of energy to the suspension, e.g., sonication, or extrusion of the vesicles through a French pressure cell (French Press) or through a porous filter of the appropriate pore size, will convert the multilamellar sterol vesicles to unilamellar vesicles.
In order to entrap a water-soluble compound, a partially water-soluble compound or a water-insoluble compound in the sterol vesicles of the present invention, a number of approaches are possible. Compounds which either partition into the sterol bilayers (e.g., water-insoluble compounds) or water-soluble compounds may be added to the aqueous phase before formation of the vesicles in order to entrap the agent within the vesicles during formation. Alternatively, compounds which are water-insoluble or lipid soluble may be added to the suspension of sterol vesicles after the vesicles are formed, in which case the compound partitions into the sterol bilayers. In another embodiment, a water-soluble compound and the salt-form of an organic acid derivative of a sterol may be added to an organic solvent so that both are solubilized (co-solubilized). The organic solvent may then be evaporated leaving a film containing a homogeneous distribution of the water-insoluble compound and the sterol derivative. Sterol liposomes entrapping the water-insoluble compounds are formed when an aqueous buffer is added to the film with shaking.
The sterol liposomes of the present invention are particularly advantageous when used to entrap water-insoluble bioactive agents or those that are sparingly soluble in water. This enables the administration in vivo of water-insoluble drugs; and furthermore, it allows for the administration in vivo of high concentrations of the water insoluble compounds, because it allows alteration of the dose:volume ratio. The sterol vesicles of the present invention offer similar advantages when used to entrap water soluble bioactive agents. In addition, the sterol vesicles of the present invention may be used in diagnostic assays in vitro.
The present invention affords a number of advantages in that the sterol vesicles:
(1) are formed easily and rapidly;
(2) have high encapsulation efficiencies as compared with phospholipid MLVs;
(3) do not require the use of organic solvents for their preparation (although the sterol vesicles of the present invention can be prepared using organic solvents); and
(4) can entrap a bioactive or pharmaceutical agent, which when administered in vivo, is released and metabolized. The fate of the entrapped agent in vivo depends upon the mode of administration.
The present invention is further directed to a composition comprising the tris(hydroxymethyl)aminomethane salt of cholesteryl hemisuccinate and an antifungal compound, particularly when the anti-fungal agent is miconazole, terconazole or econazole, isoconazole, tioconazole, bifonazole, clotrimazole, ketoconazole, butaconazole, itraconazole, oxiconazole, fenticonazole, nystain, naftifine, amphotericin B, zinoconazole or ciclopirox olamine. The composition can be used to treat a fungal infection and can be administered topically including orally or intravaginally.
The present invention includes a composition comprising the tris(hydroxymethyl)aminomethane salt of cholesteryl hemisuccinate and a peptide, particularly a hydrophobic peptide, human growth hormone, bovine growth hormone, porcine growth hormone or insulin. The composition can be administered to increase milk production or to increase or initiate growth of a mammal.